StemTAG Alkaline Phosphatase Staining and Activity Assay Kit (Fluorometric)

Transcription

1 Product Manual StemTAG Alkaline Phosphatase Staining and Activity Assay Kit (Fluorometric) Catalog Number CBA X 100 assays FOR RESEARCH USE ONLY Not for use in diagnostic procedures

2 Introduction Embryonic stem (ES) cells are continuous proliferating stem cell lines of embryonic origin first isolated from the inner cell mass (ICM). Two distinguishing features of ES cells are their ability to be maintained indefinitely in an undifferentiated state and their potential to develop into any cell within the body. Based on previous methods developed for mouse ES cells, human ES cell lines were first established by Dr. James Thomson and colleagues. Like mouse ES cells, human ES cells express high levels of membrane alkaline phosphatase (AP) and Oct-4, a transcriptional factor critical to ICM and germline formation. However, unlike mouse ES cells, hes cells do not express stage-specific embryonic antigen (SSEA-1). In addition, prolonged propagation of hes cells is typically achieved by coculture with primary mouse embryonic fibroblasts (MEFs) serving as feeder cells. Human ES cell lines are not able to maintain their undifferentiated state in the absence of supporting feeder layer cells, even when exogenous cytokines such as leukemia inhibitory factor (LIF) and gelatin-coated plates are used. Marker Name Mouse ES Mouse EG Human ES Human EG Human EC AP SSEA-1 SSEA-4 TRA-1-60 TRA-1-81 Oct-4 unknown ES Cell = Embryonic stem cell EG Cell = Embryonic germ cell EC Cell = Embryonic carcinoma cell Table 1. Comparison of Mouse and Human Pluripotent Stem. Although stem cells from different origins require different growth conditions for self-renewal and display different cell surface markers (see Table 1), AP is the most widely used stem cell marker. The StemTAG Alkaline Phosphatase Staining and Activity Assay Kit (Fluorometric) provides an efficient system for monitoring ES cell undifferentiation/ differentiation through AP activity by both immunocytochemistry staining and quantitative activity assay.. The fluorometric alkaline phosphatase activity assay is 50 times more sensitive than that obtained with the chromogenic substrate (see CBA- 301). Related Products 1. CBA-300: StemTAG Alkaline Phosphatase Staining Kit 2. CBA-301: StemTAG Alkaline Phosphatase Activity Assay Kit (Colorimetric) 3. CBA-302: StemTAG Alkaline Phosphatase Staining and Activity Assay Kit (Colorimetric) 4. CBA-308: StemTAG Alkaline Phosphatase Staining and Activity Assay Kit (Fluorometric) 5. CBA-315: JK1 Feeder 2

3 6. CBA-312: MEF Feeder (Puromycin-resistant) 7. CBA-316: SNL Feeder 8. CBA-325: StemTAG Stem Cell Colony Formation Assay 9. CBA-320: CytoSelect 96-Well Hematopoietic Colony Forming Cell Assay Kit Components Fixing Solution (Part No. C30001): One bottle 50 ml StemTAG AP Staining Solution A (Part No. C30002): One amber bottle 20 ml StemTAG AP Staining Solution B (Part No. C30003): One amber bottle 20 ml StemTAG AP Fluorometric Substrate (100X) (Part No ): One amber tube 50 µl in DMSO Cell Lysis Buffer (Part No. C30005): One bottle 20 ml Stop Solution (Part No ): One bottle 10 ml Reference Standard (Part No ): One amber tube 100 µl of 5 mm Sodium Fluorescein in PBS Materials Not Supplied 1. Human or Mouse Embryonic Stem and Culture Medium 2. 1X PBS 3. Deionized Water 4. 1X PBST (1X PBS containing 0.05% Tween-20) 5. Light Microscope 6. Fluorescence microplate reader equipped with a 480 nm excitation filter and 520 nm emission filter Storage Store StemTAG AP Fluorometric Substrate (100X) protected from light at -20ºC. Store all other components at 4ºC. Preparation of Reagents StemTAG AP Staining Solution: Prepare FRESH 1X StemTAG AP Staining Solution by mixing equal volume of StemTAG AP Staining Solution A and StemTAG AP Staining Solution B. The volume of StemTAG AP Staining Solution needed is based on the number of samples. The chart below is suggested for samples in a 24-well plate, and may be modified accordingly to suit other culture plate sizes. 3

4 Reagents Half plate (12 samples) 1 plate (24 samples) 4 plates (96 samples) Staining Solution A 2.4 ml 4.8 ml 9.6 ml Staining Solution B 2.4 ml 4.8 ml 9.6 ml Total 4.8 ml 9.6 ml 19.2 ml Table 2. Preparation of StemTAG AP Staining Solution. 1X Substrate Solution: Freshly prepare only enough for immediate applications. Prepare a 1X Substrate Solution by diluting the provided 100X stock 1:100 in deionized water. Keep the 1X Substrate Solution away from light. Preparation of Standard Curve 1. Prepare a 1:5 dilution series of Reference Standards in the concentration range of 0 50 µm by diluting the 5 mm Reference Standard stock in 1X PBS (see Table 2). Standard Tubes Reference Standard (µl) 1XPBS (µl) Fluorescein (nm) , of Tube # , of Tube # , of Tube # of Tube # of Tube # of Tube # Table 3. Preparation of Fluorescein Reference Standards 2. Transfer 100 µl of each reference standard to a 96-well plate suitable for fluorescence measurement. Add 50 μl of Stop Solution and mix by placing the plate on an orbital plate shaker for 30 seconds. 3. Read the fluorescence with a fluorescence plate reader at 480 nm excitation /520 nm emission. Assay Protocol: AP Staining (24-Well Plate) 1. Culture mouse ES cells in medium containing LIF; alternatively, culture human ES cells on a MEF feeder layer. 2. Gently aspirate the medium from the ES cells and wash the cells with 1 ml of 1X PBST. Aspirate the wash solution. 3. Add Fixing Solution to the cells, 0.4 ml per well for a 24-well plate. Incubate at room temperature for 2 minutes. 4. Remove the fixing solution and wash the fixed cells twice with 1 ml of 1X PBST. 4

5 5. Aspirate the final wash, and add 0.4 ml per well of freshly prepared StemTAG AP Staining Solution (see Preparation of Reagents section). 6. Incubate the cells at room temperature for minutes, protected from light. 7. Remove the AP Staining Solution, and then wash the stained cells twice with 1 ml of 1X PBS. Store cells in 1X PBS at 4ºC. For long-term storage, overlay the cells with 1X PBS containing 20% Glycerol. Store at 4ºC. 8. Count the red stained cell colonies (undifferentiated ES cells) vs. colorless colonies (differentiated ES cells) using a light microscope. Assay Protocol: AP Activity Assay 1. Culture mouse ES cells in medium containing LIF; alternatively, culture human ES cells on a MEF feeder layer. 2. Gently aspirate the medium from the ES cells and wash the cells twice with cold PBS. Aspirate the wash solutions. 3. Lyse the cells in Cell Lysis Buffer (0.5 ml for a 35 mm dish). 4. After a 10-minute incubation at 4ºC, remove the solution and spin down the cell debris at 12,000 X g for 10 minutes. Save the supernatant as cell lysate. Perform a BCA assay or other protein assay to determine the protein concentration of the cell lysate. 5. Add 50 μl of cell lysate to a 96-well plate suitable for fluorescence measurement. In addition, prepare blank wells that contain 50 μl Cell Lysis Buffer. We recommend testing samples in triplicate. 6. Initiate the reaction by adding 50 μl of 1X Substrate Solution. Mix the reaction mixture thoroughly by pipetting to ensure homogeneity. 7. Immediately begin reading sample and blank wells with a fluorescent microplate reader with an excitation wavelength of 480nm and an emission wavelength of 520nm. Read the wells in increments between 1 and 5 minutes for a total of 30 minutes. Note: To normalize data, the readings from samples must be subtracted from a blank sample (no enzyme). 8. (optional) If an end-point reading is desired, stop the reaction by adding 50 μl of Stop Solution and mixing by placing the plate on an orbital plate shaker for 30 seconds. Read the fluorescence with a fluorescence plate reader at 480 nm/520 nm. 5

6 Example of Results The following figures demonstrate typical results with the StemTAG Alkaline Phosphatase Activity Assay Kit (Fluorometric). One should use the data below for reference only. This data should not be used to interpret actual results. Figure 1: AP staining of ES. Murine embryonic stem cells (ES-D3) are maintained in an undifferentiated stage on gelatin-coated dishes in the presence of LIF, as indicated by the high AP activity. To induce differentiation, LIF was withdrawn over a period of several days; various differentiation events were observed (cells became flattened and enlarged with reduced proliferation). At the end of day 5, AP staining of undifferentiated cells was performed with the StemTAG Alkaline Phosphatase Staining Kit (Cat # CBA-300). 6

7 Figure 2: Fluorescein Reference Standard Curve and AP Activity Assay. Top: A serial 5-fold dilution of Fluorescein Reference Standards was prepared, and the fluorescence was read with a fluorescence plate reader at 480 nm/520 nm. Bottom: Mouse embryonic D3 cells were grown in the presence or absence of LIF for 5 days. Cell lysates were assayed for AP activity according to the Activity Assay Instructions. References 1. Wobus AM, Holzhausen H, Jäkel P et al. (1984) Exp Cell Res 152: Thomson JA, Itskovitz-Eldor J, Shapiro SS et al. (1998) Science 282: Smith AG, Nichols J, Robertson M et al. (1992) Dev Biol 151: Reubinoff BE, Pera MF, Fong CY et al. (2000) Nat Biotechnol 18:

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